This invention relates generally to the field of measuring an analyte in a liquid medium. More specifically, it relates to an immunoassay for the measurement of an analyte in a biological sample.
Boguslaski, R. C. et al., U.S. Pat. No. 4,134,792 (1979) describe the use of a reversibly binding enzyme modulator as a labeling substance for the detection of an analyte in a liquid medium, and in particular, the use of competitive inhibitors coupled to an analog of the analyte in immunoassays.
Dorn, A. R., U.S. Ser. No. 09/328,741 filed Jun. 9, 1999 describes a method for the enzymatic measurement of mycophenolic acid in a biological sample.
Inosine-5xe2x80x2-monophosphate dehydrogenase (EC 1.1.1.205) catalyzes the NAD-dependent oxidation of inosine-5xe2x80x2-monophosphate (IMP) to xanthosine-5xe2x80x2-monophosphate (XMP), Magasanik, B. et al., J. Biol. Chem. 226:339-350 (1957) and Jackson et al., Nature 256:331-333 (1975). The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release. First, IMP binds to IMPDH. This is followed by the binding of the cofactor NAD. The reduced cofactor, NADH, is then released from the product, followed by the product, XMP. This mechanism differs from that of most other known NAD-dependent dehydrogenases, which have either a random order of substrate addition or require NAD to bind before the substrate.
Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced, Collart et al., J. Biol. Chem. 263:15769-15772 (1988) and Natsumeda et al., J. Biol. Chem. 265:5292-5295 (1990). Each isoform is 514 amino acids, and both isoforms share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa, Yamada et al., Biochemistry 27:2737-2745 (1988).
In its narrowest application, the present invention is exemplified by a quantitative homogeneous immunoassay specific for mycophenolic acid based upon the specific uncompetitive inhibition of the enzyme inosine-5xe2x80x2-monophosphate dehydrogenase (IMPDH) by mycophenolic acid. IMPDH inhibition depends only upon the concentration of mycophenolic acid due to the uncompetitive nature of inhibition by mycophenolic acid. Thus, the greater the mycophenolic acid concentration, the greater the inhibition of the enzyme. Measuring the formation of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm monitors the reaction. IMPDH catalyzes the following reaction: 
Development of a homogeneous immunoassay can be accomplished through the attachment of a ligand to a position on mycophenolic acid or an MPA derivative or any uncompetitive inhibitor of IMPDH where such attachment does not interfere with the uncompetitive inhibition of IMPDH. In the absence of analyte, analyte-specific antibody binds the ligand-MPA derivative and prevents its inhibition of IMPDH. In the presence of analyte, analyte binds to its antibody, thus freeing up ligand-MPA derivative to inhibit IMPDH (Table 3, part C.).
The rate of formation of NADH (reduced nicotinamide adenine dinucleotide) can be measured by monitoring the change in absorption at a wavelength of 340 nm, i.e., the characteristic absorption region of NADH, and this change in absorption can then be correlated to analyte concentration.
Attractive positions for ligand attachment occur on the hexanoic chain of mycophenolic acid and its derivatives, especially at the 4xe2x80x2 carbon and the 5xe2x80x2 carbon (FIG. 1). Several compounds meet these criteria. See Nelson, P. H. et al., Journal of Medicinal Chemistry 33:833-838 (1990), Rohloff, J. C.,et al., Tetrahedron Letters 36 (43):7803-7806 (1995), Artis, D. R. et al., PCT Publication WO 95/22536 (1995) and Artis, D. R. et al., PCT Publication WO 95/22538 (1995). Several of these derivatives show greater inhibition than mycophenolic acid and thus could impart greater sensitivity to a homogeneous immunoassay. See Smith, D. B. et al., J. Org. Chem. 61:2236-2241 (1996).
In a preferred embodiment of the present invention, an uncompetitive inhibitor, mycophenolic acid (MPA) was coupled to a ligand and used in a homogeneous immunoassay for mycophenolic acid.
Use of an uncompetitive inhibitor-ligand conjugate is preferred over a competitive inhibitor-ligand conjugate since the former conjugate is far less susceptible to interferences from drugs and naturally occurring substances, which commonly can be competitive inhibitors of enzymes. Uncompetitive inhibitors of enzymes are rare in nature and should therefore be less susceptible to interferences from drugs and natural substances.
In its broadest application, the present invention can be used to measure any analyte. Specifically exemplified herein is the use of the invention for measurement of mycophenolic acid, digoxigenin, thyroxine and theophylline. Also taught is the use of the invention for measurement of any analyte including drugs or drug derivatives, hormones, polypeptides and oligonucleotides. Examples of other drugs or drug derivatives which would be suitable for assay using the method of the present invention include therapeutic drugs such-as antibiotics, e.g., gentamicin, amikacin, tobramycin, netilmicin and vancomycin; cardiac drugs, e.g., digoxin, digitoxin, N-acetyl procainamide, procainamide, quinidine and lidocaine; anti-seizure drugs, e.g., phenytoin, phenobarbital, primidone, valproic acid, ethosuximide and carbamazepine; analgesics, e.g., acetaminophen and acetylsalicylic acid; and immunosuppressants, e.g., MPA, cyclosporin, rapamycin (sirolimus) and FK506 (tacrolimus), and drugs of abuse such as amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine metabolite (benzoylecgonine), methadone, opiates, phencyclidine, propoxyphene and LSD. Examples of hormones which would be suitable for assay using the method of the present invention include thyroxine, thyroid stimulating hormone, estrogen, progesterone, testosterone, prolactin, follicle stimulating hormone, chorionic gonadotropin and leuteinizing hormone. Examples of polypeptides which would be suitable for assay using the method of the present invention include proteins and epitopes such as hemoglobin Alc, troponin-T and troponin-I. Examples of oligonucleotides which would be suitable for assay using the method of the present invention include specific oligonucleotide sequences that can hybridize under stringent conditions with sequences sufficiently specific for the detection and quantification of gonorrhoeae, human immunodeficiency virus (HIV), chlamydia and hepatitis infections.
Another aspect of the present invention relates to a test kit for conducting an assay for the determination of as analyte via the method of the present invention comprising, in packaged combination, one or more reagent compositions comprising NAD, IMPDH, IMP and a conjugate comprising MPA and a ligand of the analyte.